#COVID19 diagnostics is seeing newer heights each passing day with the whole world trying in unison. So the continuing efforts of scientists has borne the '#DETECTR' (DNA Endonuclease Targeted #CRISPR Trans Reporter) assay which is based on the Cas12a protein's collateral trans-cutting activity.
FIGURE: The Cas12a based diagnostic schematic interpretation.
Principle
LAMP is performed beforehand with the 3 pairs of primers, #Bstpolymerase and a buffer system to proceed to the DETECTR assay. The DETECTR assay is based on #LAMP PCR products which can be specifically cleaved by #Cas12a protein, thereby activating its collateral cutting activity ultimately chopping fluorophore-labelled #ssDNA reporter molecules. These cut reporter molecules are lastly read out with the help of #LateralFlowDipsticks.
Protocol
LAMP Primers were designed targeting the E (envelope) and N (nucleoprotein) genes of SARS-CoV-2. Along with 3 pairs of LAMP primers targeted at N and E gene, RNaseP specific LAMP primers were also designed using PrimerExplorer v.5 software (Eiken Chemical Co).
The Cas12a specific guide RNAs were designed utilizing #ClustalOmega v.1.2.4 and #Jalview v.2.11 softwares.
•Step 1: RT reaction and Isothermal amplification (62°C, 20 min): This step converts the sample RNA into dsDNA PCR products specific to SARS-CoV2 N gene and E gene and human specific RNaseP gene.
•Step 2: Cas12 detection (37°C, 10 min): In this step Lba Cas12a from Lachnospiraceae bacterium ND2006, specific guide RNAs (gRNAs) and substrate reporter molecules are mixed and incubated at 37°C for 10 min. The trans/ collateral cleavage of reporter DNA occurs.
•Step 3: Lateral flow strip result analysis (RT, 2-3mins): Milenia HybriDetect 1 (TwistDx) lateral flow strip (compatible with FAM/Biotin) is inserted directly into reaction, until test bands (control/sample) appear in the strip.
TABLE: Interpretation of results
Really need of the hour?
1. The DETECTR assay has comparable accuracy to RTqPCR and uses routine protocols and commercially available ‘off-the-shelf’ reagents, although is less sensitive than RTqPCR.
2. Advantage over RTqPCR is integration with easy-to-use reporting formats such as lateral flow dipsticks (#LFD) and no requirement for complex laboratory infrastructure.
3. Its advantage over LAMP is more specificity, and unbiased analysis of results with LFD.
Lets hope the approval comes soon!
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